hTM Cells Research Articles

Involvement of autotaxin in the pathophysiology of elevated intraocular pressure in Posner-Schlossman syndrome

To examine whether autotaxin (ATX) in the aqueous humor causes elevated intraocular pressure (IOP) in patients with Posner-Schlossman syndrome (PSS). ATX and transforming growth factor beta (TGF-β) in the aqueous humor were quantified in PSS patients. The expression of ATX and TGF-β in cytomegalovirus (CMV)-infected-human trabecular meshwork (hTM) cells was examined. Biological changes in hTM cells and monkey Schlemm’s canal endothelial (SCE) cells cultured in the conditioned medium of CMV-infected hTM cells were analyzed. The expression of ATX and TGF-β1 was upregulated in the aqueous humor of CMV-positive PSS patients, and the level of ATX in the aqueous humor was positively correlated with IOP. CMV infection upregulated ATX and TGF-β1 in hTM cells. The conditioned medium induced fibrotic changes in hTM cells and reduced SCE permeability, which was attenuated by an ATX inhibitor, a lysophosphatidic acid receptor antagonist, and a Rho kinase inhibitor. ATX in the aqueous humor induced by CMV infection may trigger elevated IOP. Modulating ATX activity may be a novel treatment modality for PSS.

Transduction optimization of AAV vectors for human gene therapy of glaucoma and their reversed cell entry characteristics

The trabecular meshwork (TM) of the eye is responsible for maintaining physiological intraocular pressure (IOP). Dysfunction of this tissue results in elevated IOP, subsequent optic nerve damage and glaucoma, the world’s leading cause of irreversible blindness. IOP regulation by delivering candidate TM genes would offer an enormous clinical advantage to the current daily-drops/surgery treatment. Initially, we showed that a double-stranded AAV2 (scAAV2) transduced the human TM very efficiently, while its single-stranded form (ssAAV2) did not. Here, we quantified transduction and entry of single- and double-strand serotypes 1, 2.5, 5, 6, 8, and 9 in primary, single individual-derived human TM cells (HTM). scAAV2 exhibited highest transduction in all individuals, distantly followed by scAAV2.5, scAAV6, and scAAV5. Transduction of scAAV1, scAAV8, and scAAV9 was negligible. None of the ssAAV serotypes transduced, but their cell entries were significantly higher than those of their corresponding scAAV. Tyrosine scAAV2 capsid mutants increased transduction in HTM cultured cells and all TM-outflow layers of perfused postmortem human eyes. These studies provide the first serotype optimization for gene therapy of glaucoma in humans. They further reveal biological differences between the AAV forms in HTM cells, whose understanding could contribute to the development of gene therapy of glaucoma.

An injectable peptide hydrogel for reconstruction of the human trabecular meshwork

Glaucoma is a leading cause of irreversible blindness worldwide. Current treatments of glaucoma involve lowering the IOP by means of decreasing aqueous humor production or increasing non-trabecular aqueous humor outflow with the help of IOP-lowering eye drops, nanotechnology enabled glaucoma drainage implants, and trabeculectomy. However, there is currently no effective and permanent cure for this disease. In order to investigate new therapeutic strategies, three dimensional (3D) biomimetic trabecular meshwork (TM) models are in demand. Therefore, we adapted MAX8B, a peptide hydrogel system to bioengineer a 3D trabecular meshwork scaffold. We assessed mechanical and bio-instructive properties of this engineered tissue matrix by using rheological analysis, 3D cell culture and imaging techniques. The scaffold material exhibited shear-thinning ability and biocompatibility for proper hTM growth and proliferation indicating a potential utilization as an injectable implant. Additionally, by using a perfusion system, MAX8B scaffold was tested as an in vitro platform for investigating the effect of Dexamethasone (Dex) on trabecular meshwork outflow facility. The physiological response of hTM cells within the scaffold to Dex treatment clearly supported the effectiveness of this 3D model as a drug-testing platform, which can accelerate discovery of new therapeutic targets for glaucoma. Statement of significanceArtificial 3D-TM (3-dimentional Trabecular Meshwork) developed here with hTM (human TM) cells seeded on peptide-hydrogel scaffolds exhibits the mechanical strength and physiological properties mimicking the native TM tissue. Besides serving a novel and effective 3D-TM model, the MAX8B hydrogel could potentially function as an injectable trabecular meshwork implant.

The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress

ABSTRACT The trabecular meshwork (TM) is a key regulatory tissue of intraocular pressure (IOP) in the anterior chamber of eye. Dysfunction of the TM causes resistance to outflow of aqueous humor, which in turn leads to elevated IOP, a main risk factor of glaucomatous neurodegeneration. Due to variations in IOP, TM cells are continuously exposed to mechanical deformations. We previously reported activation of macroautophagy/autophagy, as one of the physiological responses elicited in TM cells following mechanical strain application. By using biochemical fractionation analysis and imaging techniques, we demonstrate here for the first time the nuclear accumulation of the autophagic marker MAP1LC3/LC3 (microtubule associated protein1 light chain 3)-II, endogenous and exogenously added (AdGFP-LC3, AdtfLC3), in response to cyclic mechanical stress (CMS). Wheat germ agglutinin (WGA) and leptomycin B treatment suggest LC3 to enter the nucleus by passive diffusion, but to exit in an XPO1/CRM1 (exportin 1)-dependent manner in human TM (hTM) cells. While blockage of nuclear export leads to accumulation of LC3 with promyelocytic leukemia (PML) bodies, nuclear LC3 localizes in the nucleolus in cells under CMS. Moreover, nuclear LC3 co-immunoprecipitated with NUFIP1, a ribosome receptor for starvation-induced ribophagy. More interestingly, we further demonstrate that NUFIP1 translocates from the nucleus to LAMP2 (lysosomal associated membrane protein 2)-positive organelles in the stretched cells without triggering ribophagy, suggesting a more general role of NUFIP1 as a selective autophagy receptor for another yet-to-be-identified target in CMS and a surveillance role of nuclear LC3 against stretch-induced damage. Abbreviation AdGFP: adenovirus encoding GFP; ATG: autophagy-related; BSA: bovine serum albumin; CMS: cyclic mechanical stretch; Co-IP: coimmunoprecipitation; DAPI: 4′,6-diamidino-2-phenylindole; DFCs: dense fibrillar components; EM: electron microscopy; FCs: fibrillar centers; GCs: granular components; GFP: green fluorescent protein; hTM: human trabecular meshwork; HBSS: Hanks balanced salt solution; IOP: intraocular pressure; LAMP1/2: lysosomal associated membrane protein 1/2; LepB: leptomycin B; MTOR: mechanistic target of rapamacyin kinase; NES: nuclear export signals; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NLS: nuclear localization signal; NPCs: nuclear pore complexes; NUFIP1: nuclear FMR1 interacting protein 1; NS: non-stretched; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; pfu: plaque-forming units; PML: promyelocytic leukemia; RFP: red fluorescent protein; RPS15A: ribosomal protein S15a; RPL26: ribosomal protein L26; rRNA: ribosomal RNA; SIRT1: sirtuin 1; SQSTM1/p62: sequestosome 1; tfLC3: mRFP-GFP tandem fluorescent-tagged LC3; TM: trabecular meshwork; WB: western blot; WDR36: WD repeat domain 36; WGA: wheat germ agglutinin; XPO1/CRM1: exportin 1.

The LRRC8-mediated volume-regulated anion channel is altered in glaucoma

Regulation of cellular volume is an essential process to balance volume changes during cell proliferation and migration or when intracellular osmolality increases due to transepithelial transport. We previously characterized the key role of volume-regulated anion channels (VRAC) in the modulation of the volume of trabecular meshwork (TM) cells and, in turn, the aqueous humour (AH) outflow from the eye. The balance between the secretion and the drainage of AH determines the intraocular pressure (IOP) that is the major casual risk factor for glaucoma. Glaucoma is an ocular disease that causes irreversible blindness due to the degeneration of retinal ganglion cells. The recent identification of Leucine-Rich Repeat-Containing 8 (LRRC8A-E) proteins as the molecular components of VRAC opens the field to elucidate their function in the physiology of TM and glaucoma. Human TM cells derived from non-glaucomatous donors and from open-angle glaucoma patients were used to determine the expression and the functional activity of LRRC8-mediated channels. Expression levels of LRRC8A-E subunits were decreased in HTM glaucomatous cells compared to normotensive HTM cells. Consequently, the activity of VRAC currents and volume regulation of TM cells were significantly affected. Impaired cell volume regulation will likely contribute to altered aqueous outflow and intraocular pressure.

Establishment of human trabecular meshwork cell cultures using nontransplantable corneoscleral rims.

Human trabecular meshwork (hTM) cell isolation in academic settings utilizes the motile nature of these cells, allowing them to migrate away from the explant and proliferate on distal regions of the culture substrate. Corneoscleral rims used for transplantation are a potential source of explants for the establishment of hTM cell cultures. However, cell isolation and the initiation of primary cell cultures from ocular tissues stored in Optisol-GS medium for an extended period of time (>6 days) has proven difficult, since Optisol-GS remarkably reduces cell viability and cellularity. Therefore, explants obtained from ocular tissues stored in Optisol-GS do not often provide adequate cell yield to initiate primary cell cultures if conventional culture techniques are used. Therefore, the majority of the research on primary hTM cell isolation has been accomplished using donor tissue obtained within 72 h postmortem. The goal of this study was to develop an hTM cell isolation procedure from nontransplantable ocular materials, utilizing the anchorage dependency of TM cells. This procedure yielded functionally viable cells, eficiently dissociated from the trabecular meshwork. Isolated cells demonstrated typical hTM cell characteristics including monolayer formation, contact inhibition, phagocytosis, and responses to glucocorticoid exposure. To the best of our knowledge, this is the first time an expired explant has been utilized in the successful isolation of hTM cells. Our results clearly demonstrate the advantage of increasing the anchor points of hTM cells for enhanced cell migration out from the explants, which have limited cell proliferative capacity.

Lysophosphatidic Acid Induces ECM Production via Activation of the Mechanosensitive YAP/TAZ Transcriptional Pathway in Trabecular Meshwork Cells.

PurposeLysophosphatidic acid (LPA), a bioactive lipid, has been shown to increase resistance to aqueous humor outflow (AH) through the trabecular meshwork (TM). The molecular basis for this response of the TM to LPA, however, is not completely understood. In this study, we explored the possible involvement of mechanosensitive Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding domain (TAZ), transcriptional activation in extracellular matrix (ECM) production by LPA-induced contractile activity in human TM cells (HTM).MethodsThe responsiveness of genes encoding LPA receptors (LPARs), LPA hydrolyzing lipid phosphate phosphatases (LPPs), and the LPA-generating autotaxin (ATX) to cyclic mechanical stretch in HTM cells, was evaluated by RT-quantitative (q)PCR. The effects of LPA and LPA receptor antagonists on actomyosin contractile activity, activation of YAP/TAZ, and levels of connective tissue growth factor (CTGF), and Cyr61 and ECM proteins in HTM cells were determined by immunoblotting, mass spectrometry, and immunofluorescence analyses.ResultsCyclic mechanical stretch significantly increased the expression of several types of LPARs, LPP1, and ATX in HTM cells. LPA and LPA receptor–dependent contractile activity led to increases in both, the protein levels and activation of YAP/TAZ, and increased the levels of CTGF, Cyr61, α-smooth muscle actin (α–SMA), and ECM proteins in HTM cells.ConclusionsThe results of this study reveal that LPA and its receptors stimulate YAP/TAZ transcriptional activity in HTM cells by modulating cellular contractile tension, and augment expression of CTGF that in turn leads to increased production of ECM. Therefore, YAP/TAZ-induced increases in CTGF and ECM production could be an important molecular mechanism underlying LPA-induced resistance to AH outflow and ocular hypertension.

A Novel Selective Soluble Guanylate Cyclase Activator, MGV354, Lowers Intraocular Pressure in Preclinical Models, Following Topical Ocular Dosing.

The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.

Baicalin alleviates oxidative stress damage in trabecular meshwork cells in vitro.

Oxidative stress and inflammation play a key role in pathophysiology of glaucoma. Baicalin is known as an anti-oxidative and anti-inflammatory substance, possessing the potential to treat glaucoma. This study was designed to assess the anti-oxidative and anti-inflammatory potentials in culture human trabecular meshwork (hTM) cells. Using hTM cells as the in vitro model, we investigated the effects of baicalin on oxidative stress-induced markers for hTM impairments. We pre-incubated hTM cells with baicalin before hydrogen peroxide stressing or pre- plus co-incubated with baicalin before and during stressing and monitored the cell death, production of intracellular reactive oxygen species (iROS) and inflammatory cytokines, accumulation of carbonylated proteins, and activity of senescence marker. Samples that received pre- plus co-treatment with 10 or 15μM baicalin showed significantly increased cell survival and decreased iROS production. Further studies demonstrated that pre- plus co-treatment with 15μM baicalin significantly inhibited proinflammatory factor IL-1α and ELAM-1 production, decreased activities of senescence marker SA-β-gal, and lowered carbonylated protein levels. In contrast, samples that received only pre-treatment did not show any of these protective effects. Baicalin can protect hTM cells against oxidative stress, shedding light on potential treatment for glaucoma.

New Insight of Common Regulatory Pathways in Human Trabecular Meshwork Cells in Response to Dexamethasone and Prednisolone Using an Integrated Quantitative Proteomics: SWATH and MRM-HR Mass Spectrometry.

The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-β pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.

Prdx6 retards senescence and restores trabecular meshwork cell health by regulating reactive oxygen species

A progressive decline in antioxidant potential and accumulation of reactive oxygen species (ROS) are major causes of pathogenesis of several diseases, including glaucoma. Trabecular meshwork (TM) dysfunction resulting in higher intraocular pressure (IOP) is a hallmark of glaucoma, but its causes are unclear. Using human (h) TM cells derived from glaucomatous and normal subjects of different ages and cells facing oxidative-stress, we showed that specific loss of moonlighting antioxidant protein Peroxiredoxin (Prdx) 6 in aging or in glaucomatous TM cells caused ROS accumulation and pathobiological changes in TM cells. Prdx6 limits the levels of ROS, thus preventing overstimulation of genes and resultant deleterious effects. We found that Prdx6 levels declined in aging and were reduced dramatically in glaucomatous and aged TM cells. Biochemical assays revealed enhanced levels of ROS, and high expression/activation of TGFβs and its responsive extracellular matrix genes α-SM, fibronectin, TGase2 and Tsp1 in aged or glaucomatous cells. Furthermore, hTM cells displayed typical features of the combined effects of TGFβs and oxidative-stress-induced cellular changes, showing increased levels of lipid peroxidation, oxidative DNA damage, and senescence markers p16, p21 and SA-βgal activity, along with reduced levels of telomerase expression and activity. Exposure to oxidative-stress (H2O2) or knocking down of Prdx6 (with antisense) accelerated this process. Importantly, Prdx6 delivery to sick or aged TM cells reversed the process. We propose Prdx6 as a potential therapeutic target to guard the TM from oxidative-stress and age-dependent accumulation of ROS by balancing redox-homeostasis to prevent ocular disorders, like glaucoma.

TRPV4 regulates calcium homeostasis, cytoskeletal remodeling, conventional outflow and intraocular pressure in the mammalian eye.

An intractable challenge in glaucoma treatment has been to identify druggable targets within the conventional aqueous humor outflow pathway, which is thought to be regulated/dysregulated by elusive mechanosensitive protein(s). Here, biochemical and functional analyses localized the putative mechanosensitive cation channel TRPV4 to the plasma membrane of primary and immortalized human TM (hTM) cells, and to human and mouse TM tissue. Selective TRPV4 agonists and substrate stretch evoked TRPV4-dependent cation/Ca2+ influx, thickening of F-actin stress fibers and reinforcement of focal adhesion contacts. TRPV4 inhibition enhanced the outflow facility and lowered perfusate pressure in biomimetic TM scaffolds populated with primary hTM cells. Systemic delivery, intraocular injection or topical application of putative TRPV4 antagonist prodrug analogs lowered IOP in glaucomatous mouse eyes and protected retinal neurons from IOP-induced death. Together, these findings indicate that TRPV4 channels function as a critical component of mechanosensitive, Ca2+-signaling machinery within the TM, and that TRPV4-dependent cytoskeletal remodeling regulates TM stiffness and outflow. Thus, TRPV4 is a potential IOP sensor within the conventional outflow pathway and a novel target for treating ocular hypertension.

Genetic association and stress mediated down-regulation in trabecular meshwork implicates MPP7 as a novel candidate gene in primary open angle glaucoma.

BackgroundGlaucoma is the largest cause of irreversible blindness affecting more than 60 million people globally. The disease is defined as a gradual loss of peripheral vision due to death of Retinal Ganglion Cells (RGC). The RGC death is largely influenced by the rate of aqueous humor production by ciliary processes and its passage through the trabecular meshwork (TM) in the anterior part of the eye. Primary open angle glaucoma (POAG), the most common subtype, is a genetically complex disease. Multiple genes and many loci have been reported to be involved in POAG but taken together they explain less than 10 % of the patients from a genetic perspective warranting more studies in different world populations. The purpose of this study was to perform genome-wide search for common variants associated with POAG in an east-Indian population.MethodsThe study recruited 746 POAG cases and 697 controls distributed into discovery and validation cohorts. In the discovery phase, genome-wide genotype data was generated on Illumina Infinium 660 W-Quad platform and the significant SNPs were genotyped using Illumina GGGT assay in the second phase. Logistic regression was used to test association in the discovery phase to adjust for population sub-structure and chi-square test was used for association analysis in validation phase. Publicly available expression dataset for trabecular meshwork was used to check for expression of the candidate gene under cyclic mechanical stress. Western blot and immunofluorescence experiments were performed in human TM cells and murine eye, respectively to check for expression of the candidate gene.ResultsMeta-analysis of discovery and validation phase data revealed the association of rs7916852 in MPP7 gene (p = 5.7x10−7) with POAG. We have shown abundant expression of MPP7 in the HTM cells. Expression analysis shows that upon cyclic mechanical stress MPP7 was significantly down-regulated in HTM (Fold change: 2.6; p = 0.018). MPP7 protein expression was also found to be enriched in the ciliary processes of the murine eye.ConclusionUsing a genome-wide approach we have identified MPP7 as a novel candidate gene for POAG with evidence of its expression in relevant ocular tissues and dysregulation under mechanical stress possibly mimicking the disease scenario.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-016-0177-6) contains supplementary material, which is available to authorized users.

Intraocular pressure reduction and neuroprotection conferred by bone marrow-derived mesenchymal stem cells in an animal model of glaucoma

IntroductionGlaucoma is a sight-threatening retinal neuropathy associated with elevated intraocular pressure (IOP) due to degeneration and fibrosis of the trabecular meshwork (TM). Glaucoma medications aim to reduce IOP without targeting the specific TM pathology, Bone-marrow mesenchymal stem cells (MSCs) are used today in various clinical studies. Here, we investigated the potential of MSCs therapy in an glaucoma-like ocular hypertension (OHT) model and decipher in vitro the effects of MSCs on primary human trabecular meshwork cells.MethodsOcular hypertension model was performed by cauterization of 3 episcleral veins (EVC) of Long-Evans male rat eyes. MSCs were isolated from rat bone marrow, amplified in vitro and tagged with quantum dot nanocrystals. Animals were distributed as 1) MSCs group receiving 5.105cells/6μl Minimum Essential Medium and 2) MEM group receiving 6μl MEM (n = 10 each). Injections were performed into the anterior chamber of 20 days-hypertensive eyes and IOP was monitored twice a week for 4 weeks. At the end of experiment, cell distribution in the anterior segment was examined in confocal microscopy on flat mounted corneas. Moreover, we tested in vitro effects of MSCs conditioned medium (MSC-CM) on primary human trabecular meshwork cells (hTM cells) using Akt activation, myosin phosphorylation and TGF-β2-dependent profibrotic phenotype in hTM cells.ResultsWe demonstrated a rapid and long-lasting in vivo effect of MSCs transplantation that significantly reduced IOP in hypertensive eyes induced by EVC. MSCs were located to the ciliary processes and the TM. Enumeration of RGCs on whole flat-mounted retina highlighted a protective effect of MSCs on RGCs death. In vitro, MSC-CM promotes: (i) hTM cells survival by activating the antiapoptotic pathway, Akt, (ii) hTM cells relaxation as analyzed by the decrease in myosin phosphorylation and (iii) inhibition of TGF-β2-dependent profibrotic phenotype acquisition in hTM cells.ConclusionsMSCs injection in the ocular anterior chamber in a rat model of OHT provides neuroprotective effect in the glaucoma pathophysiology via TM protection. These results demonstrate that MSCs constitute promising tool for treating ocular hypertension and retinal cell degeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0168-0) contains supplementary material, which is available to authorized users.

Viral Vector Effects on Exoenzyme C3 Transferase-Mediated Actin Disruption and on Outflow Facility.

Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors. Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 μg/mL C3cs or with 5 μg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 μg C3rec. The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours. Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.

Altered Expression Levels of MMP1, MMP9, MMP12, TIMP1, and IL-1β as a Risk Factor for the Elevated IOP and Optic Nerve Head Damage in the Primary Open-Angle Glaucoma Patients.

The aim of presented work was to analyze the impact of particular polymorphic changes in the promoter regions of the -1607 1G/2G MMP1, -1562 C/T MMP9, -82 A/G MMP12, -511 C/T IL-1β, and 372 T/C TIMP1 genes on their expression level in POAG patients. Blood and aqueous humor samples acquired from 50 patients with POAG and 50 control subjects were used for QPCR and protein levels analysis by ELISA. In vivo promoter activity assays were carried on HTM cells using dual luciferase assay. All studied subjects underwent ophthalmic examination, including BCVA, intraocular pressure, slit-lamp examination, gonioscopy, HRT, and OCT scans. Patients with POAG are characterized by an increased mRNA expression of MMP1, MMP9, MMP12, and IL-1β genes as compared to the control group (P < 0.001). Aqueous humor acquired from patients with POAG displayed increased protein expression of MMP1, MMP9, MMP12, and IL-1β compared to the control group (P < 0.001). Allele -1607 1G of MMP1 gene possesses only 42,91% of the -1607 2G allele transcriptional activity and allele -1562 C of MMP9 gene possesses only 21,86% of the -1562 T allele. Increased expression levels of metalloproteinases can be considered as a risk factor for the development of POAG.

Morphological changes and viability of primary cultured human ocular trabecular meshwork cells after exposure to air

PurposeTo investigate the possible toxic effect of air exposure for an in vitro model of primary human ocular trabecular meshwork cells (HTM). MethodsHTM were isolated from five donor eyes and cultivated at 37°C. After reaching confluence the cells were seeded on two well chamber slides. The chamber slides were turned upside down in a Petri culture dish full of culture medium and filled with air using a 5ml syringe, starting this way the exposure of the cells to the air. Subsequently they were placed in the incubation chamber at 37°C. Six groups of HTM cultures were set up: group 1 consisted of samples in which HTM were exposed to air for 30min, group 2 for 1h, group 3 for 3h, group 4 for 6h, group 5 for 12h and group 6 for 24h. ResultsAt 3h after exposure, the morphology of the cells was still intact, at 6h few cells appeared deformed and exhibited characteristics of more senescent cells. At 12h after exposure to air the HTM cells started losing their typical morphology and appeared enlarged and compromised. Viability was superior to 94% in groups 1–3 while for groups 4, 5, 6 it was 82.7%, 39.5% and 12.7% respectively. ConclusionThe toxic effect of air exposure for the studied in vitro model of HTM is not significant for the time period of one to three hours. However it starts reducing viability and alternating morphology 6h after exposure until the time period of 24h, where the percentage of living cells is drastically decreased. Therefore, we suggest that the use of an air bubble especially in glaucomatous patients should be applied with caution.

Molecular Basis for Involvement of CYP1B1 in MYOC Upregulation and Its Potential Implication in Glaucoma Pathogenesis

CYP1B1 has been implicated in primary congenital glaucoma with autosomal recessive mode of inheritance. Mutations in CYP1B1 have also been reported in primary open angle glaucoma (POAG) cases and suggested to act as a modifier of the disease along with Myocilin (MYOC). Earlier reports suggest that over-expression of myocilin leads to POAG pathogenesis. Taken together, we propose a functional interaction between CYP1B1 and myocilin where 17β estradiol acts as a mediator. Therefore, we hypothesize that 17β estradiol can induce MYOC expression through the putative estrogen responsive elements (EREs) located in its promoter and CYP1B1 could manipulate MYOC expression by metabolizing 17β estradiol to 4-hydroxy estradiol, thus preventing it from binding to MYOC promoter. Hence any mutation in CYP1B1 that reduces its 17β estradiol metabolizing activity might lead to MYOC upregulation, which in turn might play a role in glaucoma pathogenesis. It was observed that 17β estradiol is present in Human Trabecular Meshwork cells (HTM) and Retinal Pigment Epithelial cells (RPE) by immunoflouresence and ELISA. Also, the expression of enzymes related to estrogen biosynthesis pathway was observed in both cell lines by RT-PCR. Subsequent evaluation of the EREs in the MYOC promoter by luciferase assay, with dose and time dependent treatment of 17β estradiol, showed that the EREs are indeed active. This observation was further validated by direct binding of estrogen receptors (ER) on EREs in MYOC promoter and subsequent upregulation in MYOC level in HTM cells on 17β estradiol treatment. Interestingly, CYP1B1 mutants with less than 10% enzymatic activity were found to increase the level of endogenous myocilin in HTM cells. Thus the experimental observations are consistent with our proposed hypothesis that mutant CYP1B1, lacking the 17β estradiol metabolizing activity, can cause MYOC upregulation, which might have a potential implication in glaucoma pathogenesis.

Kappa Opioid Receptor Localization and Coupling to Nitric Oxide Production in Cells of the Anterior Chamber

The present study was designed to determine whether kappa opioid receptors (KORs) are localized to cells of the inflow and outflow pathways of the eye and if activation of these receptors has an effect on nitric oxide (NO) production, because these effects could play a role in KOR agonist-mediated reduction of IOP. Human nonpigmented ciliary epithelial (NPCE) and trabecular meshwork (HTM-3) cells were treated with spiradoline (SPR), a selective KOR agonist, or estradiol, for 24 hours. Some cells were pretreated with the selective KOR antagonist norbinaltorphimine (norBNI) or the nonselective NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) for 30 minutes, followed by the addition of SPR. Immunofluorescent localization of KORs was determined in isolated rabbit iris-ciliary bodies (ICBs) and NPCE and HTM-3 cells. Immunohistochemical data show the localization of KORs to the rabbit ICB and more specifically to the ciliary epithelial layer. KORs were also found on cell membranes of NPCE and HTM-3 cells. Treatment of both these cell types with spiradoline caused concentration-dependent increases in the release of NO. Spiradoline-induced release of NO from both cell types was inhibited by pretreatment with norBNI and L-NAME. Results from this study show the presence of KORs on rabbit ICBs and also on NPCE and HTM cells. Activation of these KORs on both cell types resulted in KOR-mediated increases in NO production. These findings provide evidence that previously demonstrated KOR-mediated reduction in IOP could be caused, in part, by NO production in both the ciliary body and the trabecular meshwork.

MicroRNA‐24 regulates the processing of latent TGFβ1 during cyclic mechanical stress in human trabecular meshwork cells through direct targeting of FURIN

Cyclic mechanical stress (CMS) leads to alterations of cellular functions in the trabecular meshwork (TM), including the up-regulation of transforming growth factor beta 1 (TGFβ1), that can potentially contribute to the pathogenesis of glaucoma. Although microRNAs (miRNAs) are known to play important roles in many biological functions, little is known about their potential involvement in the cellular responses elicited by mechanical stress. Here we analyzed changes in miRNA expression induced by CMS, and examined the possible role of miR-24 in the response of human TM cells to CMS. CMS induced the expression of miR-24 that led to the down regulation of the subtilisin-like proprotein convertase FURIN, which is known to play a major role in the processing of TGFβ1. FURIN was confirmed as a novel target of miR-24 by 3' UTR luciferase assay and western blot. Overexpression of miR-24 resulted in a significant decrease in activated TGFβ1. This effect was mimicked by down regulation of FURIN by siRNA. Conversely, inhibition of miR-24 expression with a specific antagomir led to a small but significant increase in TGFβ1. Furthermore, the increase in active TGFβ1 induced by CMS in HTM cells was prevented by miR-24. Altogether, our results suggest that miRNAs might contribute to the regulation of responses to CMS in TM cells. Specifically, miR-24 might play an important role in modulating the induction of TGFβ1 mediated by CMS through direct targeting of FURIN.